The principal goal of this project is to extend our understanding of the mechanisms of enzymes of particular relevance to the nervous system.[unreadable] [unreadable] A study of sodium, potassium pump (Na,K-ATPase) and calcium pump (SERCA ATPase) mechanisms, in collaboration with J. Froehlich (U. Md.), is currently directed at testing a hypothesis that certain of our transient kinetic observations reflect oligomeric interactions of multiple pump molecules during their catalytic cycle. A dependent hypothesis is that such oligomeric interactions contribute to the efficiency of cation transport. Our current evidence suggests that these interactions mediate "out-of-phase" free energy transfers between two or more catalytic subunits, and that these interactions may be a general property of P-type cation pumps examined in their native state. We have recently examined the effects of the small intrinsic membrane protein, phospholamban(PLB), on the transient kinetics and oligomeric state of the SERCA1 and SERCA2a calcium pumps. PLB interacts with both the isoforms SERCA1 and SERCA2a, but is normally co-expressed only with SERCA2a, the cardiac muscle isoform. Activation of SERCA2a by beta-1-agonists increases the efficiency of calcium transport and elevates cardiac sarcoplasmic reticulum calcium stores. This involves PKA-dependent phosphorylation of PLB. PLB-pump interaction decreases the Vmax and the apparent calcium affinity of the pumps, whereas phosphorylation of PLB reverses both effects. Our data suggest that the increased pump efficiency of SERCA2a by beta-1 agonists results from a phosphorylation-induced dissociation of PLB. EPR measurements of spin-labeled SERCA2a indicate that PLB-binding changes the SERCA2a from an oligomeric state to one that is functionally monomeric. Transient kinetic studies indicate that that the skeletal muscle isoform, SERCA1, is an oligomer in the absence of PLB. SERCA1 exhibits a faster turnover and higher Vmax relative to SERCA2a. Oligomeric pump interactions are hypothesized to produce the rapid phase of E2P hydrolysis in SERCA1 that is absent in SERCA2a. We tested whether removal of PLB regulation of SERCA2a converts it to a functionally oligomeric state resembling SERCA1. This was confirmed by measuring the transient kinetic properties of pump phosphorylation using recombinant SERCA2a expressed with or without PLB in insect cells. In parallel experiments the Mahaney lab has found that spin-labeled SERCA2a co-expressed with PLB has a smaller rotational correlation time (63 ms) than either SERCA2a sans PLB (78 ms) or SERCA2a + phosphorylated-PLB (97 ms. The rotational correlation times suggest a more compact structure for SERCA2a-PLB than for SERCA2a sans PLB or SERCA2a + phosphorylated-PLB (Mahaney et al, 2005).[unreadable] [unreadable] We have also collaborated with the Neuronal Cytoskeletal Section of this laboratory to examine the kinetics of cdk5 kinase activation and inhibition. The interaction of this kinase with its different activator proteins and fragments thereof produce complex effects on its biological activity. We have described a fragment (CIP) of the cdk5-activator protein, p35, that produces high-affinity inhibition of cdk5 by selective competition with the p25 activator protein. The p25 form of the cdk5 activator appears in neurons under stress, induces hyperphosphorylation of the microtubule associated protein, tau, and transfection with the inhibitor peptide can prevent this in neuronal cell culture assays (Zheng et al, 2005). [unreadable] [unreadable] [unreadable] We are currently engaged in kinetic studies to further define the mechanism of this selective inhibition. Cdk5 is a member of the cyclin-dependent kinase family. Although cdk5 activator proteins are not cyclins, the activator interfaces with the kinases in this family are largely similar and they induce similar conformational transitions. Thus we are also exploring computational means to obtain information about the interactions of CIP and related inhibitors with the cdk5 kinase.